**2. Methods**

A search for studies published before January 2020 was performed in the PubMed and Embase databases. All authors participated in the search process. We looked for the terms "irritable bowel syndrome", "microbiota", "treatment", "prebiotic", "probiotic", "synbiotic", "FODMAP", "meta-analysis", "randomized", "clinical", "bifidobacteria", "lactobacillus", "firmicutes", "bacteroidetes", "methane", "methanogen", "diet", "fecal transplantation", "bacteriophage", and "fungi", mainly focusing on the literature that describes effects on microbiota, clinical studies, and therapeutic effects in IBS. The search was restricted to articles in the English language.

#### **3. The Microbiome in IBS**

A rash of research activity in this field from the last decade has led to the evidence that a disruption in the biodiversity, richness, and composition of the gu<sup>t</sup> microbiota—a process named dysbiosis—plays a key function in the pathogenesis of IBS [26]. Dysbiosis can take place through several mechanisms: the overgrowth or vanishing of specific bacteria species, alterations in the relative richness of bacteria, and lastly, by mutation or gene transfer [27].

In IBS patients, GI dysbiosis has been associated with a visceral increased perception of pain and enhanced mucosal permeability that is provoked by the defective mucosal epithelial barrier, interfering with gu<sup>t</sup> immune homeostasis and subsequently promoting gu<sup>t</sup> inflammation and enhancing cellular and mucosal immune activation [28,29]. Moreover, it has also been associated with changing gu<sup>t</sup> motility, low-grade chronic inflammation, alterations of the enteric nervous system, and vagal afferents neurons as well as brain functions [30–32]. In opposition, gu<sup>t</sup> microbiota can be affected by the brain activity on intestinal motility, secretion, and immune function, generating the microbiota–gut–brain axis [12,33].

Microbiological and infectious bases of IBS pathogenesis have been previously established by several groups. Halvorson et al. reported a seven-fold increased risk of post-infectious IBS after acute infectious gastroenteritis [34]. Moreover, therapeutic interventions that manipulate the gu<sup>t</sup> microbiota such as antibiotics, prebiotics, probiotics, and fecal microbial transplantation have been linked to improvements in IBS symptoms [35–37]. Several studies aiming to characterize and map the microbiome signature of IBS have shown divergent results. Nonetheless, data sugges<sup>t</sup> that there is a relative richness of proinflammatory bacterial species containing Enterobacteriaceae, with a parallel decline in *Lactobacillus* and *Bifidobacterium* [38,39]. A decreased percentage of *Lactobacillus* and *Bifidobacterium* species has also been described in the IBS microbiota, leading to disturbances in short-chain fatty acid production and in immunologic and bactericidal activity, with a negative effect on microbiota function and stability [40–46]. Interestingly, the Firmicutes/Bacteroidetes ratio is a possible indicator of bacterial population shifts, and both high and low ratios of Firmicutes/Bacteroidetes have been reported in IBS patients [47–50]. These contrasts may be explained by differences in technical methods and subtypes of IBS, as well as the severity of IBS [38]. Several groups examined gu<sup>t</sup> microbiota in different subtypes of IBS and compared the microbiota texture between different IBS subtypes as well as different IBS symptoms [48,51,52]. A study by Ringel-Kulka et al. using fecal samples from 60 patients with IBS and 20 healthy controls revealed major variances in the microbiota between the different subtypes of IBS based on clinical symptoms of abdominal bloating and bowel habits (IBS-D, IBS-C, or IBS-M type) [53]. A study examining both fecal and colonic mucosal microbiota in patients with chronic constipation found that their mucosal microbiota differed from those of the controls, with a higher abundance of Bacteroidetes species in the patients than in the controls. However, although the profile of the colonic mucosal microbiota discriminated between these two cohorts with a high level of accuracy, this finding was independent of colonic transit time. In contrast, the profile of the fecal microbiota was associated with colonic transit, but not with the clinical diagnosis of constipation [54]. Putting all of this together, numerous evidence is emerging regarding the link between microbiota and IBS pathogenesis, making microbiota manipulation strategies an attractive option for IBS management.

#### **4. Therapeutic Interventions for Microbiome Manipulation in IBS**
